Human embryonic and fetal hematopoiesis occurs in stages as several anatomical sites become transiently active. It has been accepted for almost three decades that the first phase of angio-hematopoiesis occurs around 16 days of development in the extra-embryonic niche provided by the yolk sac. The hematopoietic output of the yolk sac is gradually replaced by sequential intra-embryonic sites. Cells resembling primitive hematopoietic progenitors are first detected in the human aorta-gonad-mesonephros (AGM) region at day 27 and the population quickly expands until their disappearance by day 40. The embryonic liver is the next hematopoietic tissue to become active around weeks 5-6 of gestation, peaking at the end of the second trimester and gradually diminishing throughout the remainder of fetal development. Finally, early in the second trimester of gestation, the fetal bone marrow (BM), which will be active for the rest of life, begins to function. Until very recently, no other hematopoietic region was known to be part of the orchestrated and site-specific hematopoiesis occurring during the embryonic and fetal phases of human development. The recent discovery that the mouse placenta contains a sizable pool of hematopoietic stem cells (HSCs) at about the same time that the AGM is active, alters the current model of embryonic hematopoiesis. The placenta is a transient fetal organ that develops in advance of the embryo, which it supports by performing a myriad of transport functions. The hematopoietic and angiogenic potential of the human placenta are currently unknown. Accordingly, the goal of this proposal is to test the hypothesis that HSCs are present in the human placenta. Our preliminary data support this hypothesis. Specifically, we detected by fluorescent activated cell sorting (FACS) a population with the phenotypic characteristics of primitive progenitors (CD34++CD45low cells) that expresses several antigens that are typical of HSCs at first and second trimester placentas as well as at term. When they were purified by cell sorting and tested in hematopoietic progenitor assays, they generated colony forming unit cultures. These findings led us to propose the following experiments. In Aim 1, we will determine the frequency (by flow cytometry) and anatomical location (by immunolocalization) of putative HSCs in human placentas as a function of gestational age. We hypothesize that the frequency will decrease as pregnancy advances and their location will help us to establish their relationship to the emerging vascular system. In Aim 2, we will functionally characterize putative human placental HSCs. The goal of these experiments is to assess their hematopoietic and angiopoietic potential by isolating the candidate HSCs to a high degree of purity by cell sorting, and assess their growth potential using a battery of in vitro and in vivo assays. To further support the immature nature of the placental putative HSCs, we will determine if they express transcription factors that are crucial for hematopoiesis including SCL/tal1, GATA family members and runx 1. Project narrative: We think that this work is important because a better understanding of placental hematopoiesis could have a large impact on the field of hematopoietic stem cells (HSC) banking and transplantation. Although HSC transplantation is a critical component of medical therapy for the treatment of hematological and non- hematological pathologies, shortages in HSCs from human leukocyte antigen (HLA)-matched donors continue to limit their use and drives efforts to expand sources. The possibility of using placentas that are routinely discarded after delivery for the isolation of HSCs is exciting as this transient human organ may be an easily available and valuable source of cells that would allow significant expansion of existing banks, an especially important issue with regard to transplantation of non-Caucasian recipients.